Experiments can sometimes lead to the discovery of completely unanticipated
phenomena. Such is the case with the remarkable behavior exhibited by peptide
nanostructures (in the form of supramolecular filaments) observed during experiments
carried out by researchers from Northwestern University at beamline 5-ID of
the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) Synchrotron
Research Center at the U.S. Department of Energy's Advanced Photon Source (APS)
at Argonne National Laboratory.

A drawing depicting a bundle of 10-nanometer-diameter peptide filaments positioned in a hexagonal array. Similar phenomena may occur naturally in the cytoskeletons of cells, the cornea of the eye, and other areas of biology. Inset (at lower right) depicts the molecular structure of individual filaments. (Image courtesy of S.I. Stupp.)

According to Professor Samuel Stupp, lead author of the study recently published
in Science, while “trying to elucidate the hierarchical organization of
peptide nanostructures” his team discovered that when dispersed in water,
those filamentary nanostructures could organize into hexagonally-packed bundles.
The researchers were surprised to find that at sufficiently high concentrations
in solution, the filaments could spontaneously self-assemble into crystalline
structures (the hexagonally-packed bundles). Even more surprising was the finding
that the x-rays used to probe the nanostructures also sometimes triggered filament
crystallization. This work may impact our understanding of nanostructures in
biological systems and our ability to control the structure of materials.

The filaments used for this research possessed diameters of around 10 nanometers
and lengths on the order of tens of micrometers. The filaments were derived
from a synthetic molecule containing a short peptide sequence. Peptides are
compounds containing two or more amino acids. Here, the peptide sequence consisted
of six alanine amino acid molecules bonded to three glutamic acid molecules
- abbreviated Ala6Glu3 - which in turn was grafted to an alkyl molecule. The
resulting “supramolecules” self-assembled in water to form the filaments.

A sequence of experiments was designed to reveal the arrangement of the filaments
dispersed in water. Different aqueous concentrations of the filaments were placed
inside tiny 2-mm-diameter quartz capillaries and studied utilizing small-angle
x-ray scattering (SAXS) at the DND-CAT beamline. The concentrations ranged from
0.5 to 5 weight percent. The SAXS data revealed that all concentrations of filaments
aggregated into bundles exhibiting a hexagonal packing (see Fig. 1). The organization
of the filaments into hexagonally-packed bundles (i.e., crystallization) is
quite remarkable. But even more remarkable was the observation that the higher
concentration of filaments (2 and 5 weight percent) spontaneously crystallized,
while the lower-concentration solutions (0.5 and 1 weight percent) crystallized
only through x-ray exposure.

According to Prof. Stupp, the crystallization of the filaments, either by self-assembly
or by x-ray exposure, constitute phenomena that “we have not seen before”
in other supramolecular systems. Stupp also observed that “in doing the
experiments at the APS synchrotron, we were surprised to find that x-rays could
promote crystallization.”

A fascinating feature of the x-ray-induced crystallization was the reversibility
of the process, which was actually visible. Using the 1 weight percent solution,
a cumulative 200 seconds of x-ray irradiation turned the initially-transparent
solution opaque, indicating crystallization. After x-ray cessation, the solution’s
opacity slowly decreased until it was clear again within about 40 minutes, indicating
a return to disorder. A follow-up SAXS experiment exposed the solution to a
number of 4-second x-ray bursts. The experimental data showed that the initially-unordered
filaments (revealed by the first 4-second exposure) gradually underwent a change
to hexagonally-ordered bundles of filaments as recorded during the last x-ray
exposures. When the experiment was repeated two hours later, the SAXS data revealed
the filaments were once again disordered - the crystalline structure had disappeared.

The researchers considered whether extraneous factors might have contributed
to filament ordering. Intense x-rays can create new chemical compounds within
a solution due to ionization, as well as produce subtle heating. However, subsequent
tests of the filamentary solutions showed that neither unwanted chemical species,
nor thermal effects, had played a part in either the spontaneous or x-ray-triggered
crystallizations.

Concerning the basic mechanism responsible for crystallization, the researchers
envision that the long-term stability of the crystalline domains is a balance
between two opposing tensions: electric charges residing on the filaments (either
native or induced by x-ray irradiation) tend to push filamentary bundles apart,
while entrapment of filaments within the larger network leads to an inward mechanical
compression.

Experimental data revealed that as filament concentration grew, the number
of filaments within bundles increased as well, until a critical concentration
of filaments resulted in their spontaneous hexagonal arrangement within the
bundles (i.e. crystallization). On the other hand, lower filamentary concentrations
- unable to spontaneously crystallize - could only do so when x-rays increased
the charge density on the filaments’ surfaces, thereby changing the balance
of inter-filament forces in favor of crystallization.

The same mechanism that created their man-made crystalline filamentary networks
may well be at work in biological cells, leading Prof. Stupp to observe that
“this research could help us understand the organization of nanostructures
in biological systems, and may also have applications in controlling the structure
of materials.”